CDPKs in immune and stress signaling - Molbio

CDPKs in immune and stress signalingMarie Boudsocq1 and Jen Sheen2

1 Unite de Recherche en Ge nomique Ve ge tale, INRA-UEVE UMR1165, CNRS ERL8196, 2 rue Gaston Cre mieux, 91057 Evry cedex,

France2 Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital,

Department of Genetics, Harvard Medical School, Boston, MA 02114, USA

Review

Ca2+ has long been recognized as a conserved secondmessenger and principal mediator in plant immune andstress responses. How Ca2+ signals are sensed andrelayed into diverse primary and global signaling eventsis still largely unknown. Comprehensive analyses of theplant-specific multigene family of Ca2+-dependent pro-tein kinases (CDPKs) are unraveling the molecular, cel-lular and genetic mechanisms of Ca2+ signaling. CDPKs,which exhibit overlapping and distinct expression pat-terns, sub-cellular localizations, substrate specificitiesand Ca2+ sensitivities, play versatile roles in the activa-tion and repression of enzymes, channels and transcrip-tion factors. Here, we review the recent advances on themultifaceted functions of CDPKs in the complex immuneand stress signaling networks, including oxidative burst,stomatal movements, hormonal signaling and generegulation.

Ca2+ sensor protein kinases in immune and stresssignaling networksDespite longstanding knowledge that Ca2+ mediates plantresponses to a wide range of developmental and environ-mental stimuli through variations of its intracellular con-centrations, the molecular, cellular and genetic linksbetween Ca2+ signatures and the multiple downstreamsignaling events are largely obscure. Calcium signaturesare defined by spatio-temporal features, including ampli-tude, frequency, duration and sub-cellular location, thatare likely to contribute to Ca2+ signaling specificity alongwith the diverse proteins able to sense and decode thesesignals [1–9]. Plants possess three main families of calciumsensors: calmodulin (CaM), calcineurin B-like (CBL) andCDPKs. Unlike CaM and CBL that must relay the Ca2+-induced conformational change to protein partners,CDPKs have the unique feature of both Ca2+ sensingand responding activities within a single protein to directlytranslate Ca2+ signals into phosphorylation events[2,6,10,11]. Recently, major progress has been made touncover the central roles of CDPKs in triggering appropri-ate and diverse downstream responses in the plant im-mune and stress signaling networks. In this review, weexamine the recent advances on the molecular activationmechanism of CDPKs, as well as their versatile roles inimmune and stress signaling, such as the regulation ofoxidative burst, cell death, stomatal movements, hormonalsignaling and gene expression.

Corresponding author: Boudsocq, M. ([email protected]).

30 1360-1385/$ – see front matter � 2012 Elsevier Ltd. All rights reserved. http://dx.d

CDPK structure, regulation and Ca2+ signal relayCDPK structure and regulation

CDPKs harbor an N-terminal variable domain, a Ser/Thrkinase domain, an auto-inhibitory junction region and aregulatory calmodulin-like domain (CaM-LD) [1,2,10,11].The four EF-hand Ca2+-binding motifs of the CaM-LD areorganized into two lobes that have distinct Ca2+ affinitiesresulting in different roles in CDPK regulation [12,13]. Inthe basal state, the intramolecular interaction between thejunction region and the catalytic center maintains thekinase in an inactive state by a pseudosubstrate mecha-nism [1,2]. The C-terminal lobe of the CaM-LD exhibitinghigh Ca2+ affinity interacts with the auto-inhibitory regionat low Ca2+ level to stabilize the structure [14]. Ca2+

binding to the low-affinity N-terminal lobe of the CaM-LD induces a conformational change that releases theauto-inhibition [1,2]. As a result, deleting both the auto-inhibitory domain and the CaM-LD generates a constitu-tively active form that constitutes a powerful tool forstudying CDPK functions in vivo [15–17].

The activation model, based on the in vitro characteri-zation of recombinant proteins, is now supported by the inplanta analysis of EF-hand-mutated variants of Arabidop-sis (Arabidopsis thaliana) AtCPK21 [18]. Interestingly, therecent crystal structure of full-length apicomplexan (Toxo-plasma gondii and Cryptosporidium parvum) CDPKs inboth apo- and Ca2+-bound forms confirms the essential roleof the N-lobe of the CaM-LD in triggering CDPK activation[19]. Importantly, multiple CDPK isoforms from soybean(Glycine max) and Arabidopsis exhibit distinct Ca2+ sensi-tivities, which is consistent with their roles in decodingdifferent Ca2+ signals [20,21]. However, several CDPKs,including Arabidopsis AtCPK13 and AtCPK23, were re-cently shown to be weakly or not sensitive to Ca2+ (Table 1)[21–23]. Despite alterations in some EF-hand motifs, theCDPKs with apparently lower Ca2+-sensitivity are able tobind Ca2+ in vitro [21], which triggers the same conforma-tional change as in canonical CDPKs [24]. Moreover, mostreported CDPK assays used generic but not specific andbiologically relevant endogenous substrates to determinethe Ca2+ sensitivity of CDPK activities, which may varywith different substrates [1,20,21]. Thus, it remains possi-ble that all CDPKs exhibit Ca2+ activation in vivo withappropriate substrates. A high-throughput screen of syn-thetic peptides has identified many potential CDPK sub-strates for further characterization [25]. Nonetheless,Ca2+ also regulates other aspects of CDPKs, such as pro-tein interactions [26,27] or sub-cellular localization [24].Thus, CDPKs can sense various Ca2+ signals through the

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Table 1. Functional characterization of Arabidopsis CPKs

Protein (synonym) Gene Sub-group Expressiona N-acylation

predictionbLocalizationc Ca2+ -depd Functionc Refs

CPK1 (AK1) At5g04870 I R, S, GC N-Myr Peroxisomes,

oil bodies

Yes SA and defense, cold [13,25,46,48,

57,60,79,87,

90]

CPK2 At3g10660 I R, P N-Myr ER and other

membranes

Yes ND [21,40,42]

CPK3 (CDPK6) At4g23650 II R, S, GC N-Myr Soluble, PM,

tonoplast

Yes Herbivore, salinity,

stomata

[21,23,44,46,

53,80,88]

CPK4 At4g09570 I R, S, P, GC – Cytosol, nucleus Yes MAMP, ABA, drought,

salinity, stomata

[17,21,39,46,

51,52,88]

CPK5 At4g35310 I R, S, GC N-Myr Membrane,

cytosol, nucleus

Yes MAMP [17,21]

CPK6 (CDPK3) At2g17290 I R, S, P, GC N-Myr Membrane,

cytosol, nucleus

Yes MAMP, ABA, drought,

salinity, stomata

[17,42,74,

80,81,83,88]

CPK7 At5g12480 III R, S, GC N-Myr-Palm PM Unclear Stomata [21,46,88]

CPK8 (CDPK19) At5g19450 III R, S, GC N-Myr-Palm PM Unclear Stomata [21,46,88]

CPK9 At3g20410 II R, S, GC N-Myr-Palm PM Yes ND [21,42,46]

CPK10 (CDPK1) At1g18890 III R, S, GC N-Myr-Palm PM Unclear ABA, drought, stomata [15,21,25,

27,88]

CPK11 (CDPK2) At1g35670 I R, S, P, GC – Cytosol, nucleus Yes MAMP, ABA, drought,

salinity, stomata

[17,21,39,51,

52,88]

CPK12 (CDPK9) At5g23580 I R, S – Cytosol, nucleus Yes ABA [72]

CPK13 At3g51850 III R, S, GC N-Myr-Palm Membrane, PM Unclear Herbivore [21,23,42]

CPK14 At2g41860 III P N-Myr-Palm ND ND ND

CPK15 At4g21940 II S N-Myr-Palm ND ND ND

CPK16 At2g17890 IV P N-Myr-Palm PM ND ND [25,45,46]

CPK17 At5g12180 II P N-Myr-Palm PM ND Pollen tube growth [36]

CPK18 At4g36070 IV P N-Myr-Palm ND ND ND

CPK19 At1g61950 II – – Membrane Yes ND [21]

CPK20 At2g38910 I P N-Myr ND ND ND

CPK21 At4g04720 II R, S, GC N-Myr-Palm PM Yes Osmotic stress,

stomata

[18,22,46,82]

CPK22 At4g04710 II R, S, GC N-Myr-Palm ND ND ND

CPK23 At4g04740 II – N-Myr-Palm PM Unclear Drought, salinity,

stomata

[22,75,82]

CPK24 At2g31500 III P N-Myr-Palm ND ND ND

CPK25 At2g35890 I P N-Myr Membrane No ND [21]

CPK26 At4g38230 I P – ND ND ND

CPK27 At4g04700 II R, S, GC N-Myr-Palm ND ND ND

CPK28 At5g66210 IV R, S, GC N-Myr-Palm PM ND ND [46]

CPK29 At1g76040 II R, S – ND ND ND

CPK30 (CDPK1a) At1g74740 III R, S, GC N-Myr-Palm Membrane Unclear ABA and abiotic

stress

[15,21]

CPK31 At4g04695 II – N-Myr-Palm ND ND ND

CPK32 At3g57530 III R, S, P, GC N-Myr-Palm Membrane,

nucleus

Unclear ABA, stomata [21,47,88]

CPK33 At1g50700 II R, S N-Myr-Palm ND ND ND

CPK34 At5g19360 II P N-Myr-Palm PM Yes Pollen tube growth [25,36]

aThe expression of CPKs in root (R), shoot (S), pollen (P) and guard cell (GC) was compiled from diverse microarray data (http://www.weigelworld.org/resources/microarray/

AtGenExpress/). Several CPKs exhibit a low expression level in all organs (–).

bThe absence (–) or presence of acylation at the N-terminus, either a myristate (N-Myr) and/or a palmitate (Palm), was predicted by TermiNator program (http://

www.isv.cnrs-gif.fr/terminator2/index.html).

cAbbreviations: ER, endoplasmic reticulum; PM, plasma membrane; ND, not determined; SA, salicylic acid; MAMP, microbe-associated molecular pattern; ABA, abscisic

acid.

dSome CPKs are clearly Ca2+-dependent for their activity (Yes) whereas others exhibit no or low calcium stimulation on general and unspecific substrates despite effective

calcium binding (Unclear). Only CPK25 lacking EF-hands is truly Ca2+-independent (No). ND, not determined.

Review Trends in Plant Science January 2013, Vol. 18, No. 1

amplitude and sub-cellular location of Ca2+ rise, but howthey may distinguish frequency and duration requiresfurther investigation, such as elucidating the molecularmechanism of CDPK deactivation.

Besides Ca2+, phosphorylation, lipids and interactionwith 14-3-3 proteins have been reported to further modulate

CDPKs in vitro [1,2,4,10,11]. Recent in vivo studies, includ-ing phosphoproteomic approaches [28,29], are startingto reveal the biological significance of these regulatorymechanisms in response to various signals. For instance,stress-dependent phosphorylations correlated with kinaseactivation have been reported for the tobacco (Nicotiana

31

http://www.weigelworld.org/resources/microarray/AtGenExpress/

http://www.weigelworld.org/resources/microarray/AtGenExpress/

http://www.isv.cnrs-gif.fr/terminator2/index.html


AtCPK3

OsCPK1

OsCPK15 AtCPK17 AtCPK34 OsCPK2 OsCPK14 OsCPK25/26

AtCPK9 AtCPK33

GmCDPKγ

McCPK1

NtCDPK1

LeCPK1

OsCPK19

HvCDPK3 AtCPK19

AtCPK15

AtCPK21

AtCPK23

AtCPK22 AtCP

K27 AtCP

K31

AtCP

K29

OsC

PK12

OsCP

K8

OsCP

K20

AtCP

K7

AtCPK

8

AtCPK14 AtCPK32 OsCPK9 AtCPK10 AtCPK30 AhCPK2

AtCPK13 OsCPK3 OsCPK16

AtCPK24

OsCPK29

OsCPK21

OsCPK22

OsCPK31

OsCPK30

OsCPK4

AtCPK16

AtCPK18 OsCPK18

AtCPK28

AtCPK25

GmCDPKβAtCPK4

AtCPK11 AtCPK12

GmCDPKα

ACPK1 O

sCPK28 Zm

CPK11

OsCPK24

OsCP

K6

AtCP

K26 At

CPK5

At

CPK6

St

CDPK

4 St

CDPK

5 OsC

PK5

OsCPK13

HvCDPK4 OsCPK7

OsCPK23

OsCPK27

OsCPK10 AtCPK1 AtCPK2 NtCDPK3 NtCDPK2 LeCDPK2

OsCPK11 OsCPK17

AtCPK20

0.1

NaCDPK4

NaCDPK5

MtCDPK1

TRENDS in Plant Science

Subgroup IV

Subgroup I

Subgroup II

Subgroup III

Figure 1. Relation tree of selected plant CDPKs. The full-length amino acid sequences of CDPKs (see supplementary material online) from Arabidopsis (At, blue), rice (Os,

pink), soybean (Gm, brown), potato (St, black), barley (Hv, purple), tobacco (Nt, green), coyote tobacco (Na, green), tomato (Le, yellow) and grapevine (ACPK1, gray), maize

(Zm, gray), alfalfa (Mt, gray), ice plant (Mc, gray) and peanut (Ah, gray) were aligned and analyzed with ClustalX and TreeView algorithms. The CDPK family is divided into

four major subgroups (I–IV). The branched lengths are proportional to divergence and the scale of 0.1 represents 10% change. The CDPKs with known biological functions

are highlighted in bold.


tabacum) NtCDPK2 and NtCDPK3 [30]. In maize (Zeamays), the lipid activation of ZmCPK11 by direct bindingof phosphatidic acid probably occurs in response to wound-ing [31].

CDPK expression and localization

CDPKs are encoded by multigene families of 34 membersin Arabidopsis [1,2,10], 31 in rice (Oryza sativa) [32,33] andat least 20 in wheat (Triticum aestivum) [34], and aredivided into four subgroups (Figure 1 and Table 1). Exten-sive transcriptomic analyses have revealed different ex-pression patterns for each isoform, which contributes to thefunctional specificity of the CDPKs [1,33–35]. SomeCDPKs are expressed in most organs whereas others arespecific to some tissues. For example, AtCPK17 andAtCPK34 are preferentially expressed in mature pollenand regulate pollen tube growth [36]. Differential expres-sion of CDPKs has been observed in response to diverse

32

stimuli, including abscisic acid (ABA), cold, drought, salin-ity, heat, elicitors and pathogens [16,33–35], which corre-lates with the presence of stress-responsive cis-elements inrice CDPK gene promoters [35]. CDPK protein accumula-tion has also been reported after cold or ABA treatment,resulting in enhanced CDPK kinase activity [37–39].

Most CDPKs have a predicted N-myristoylation siteinvolved in membrane targeting (TermiNator, http://www.isv.cnrs-gif.fr/terminator2/index.html), which hasbeen confirmed in vitro for some of them [12,40–45]. Thisirreversible co-translational acylation requires a secondpost-translational signal to maintain the membrane asso-ciation, such as reversible palmitoylation [30,41], a poly-basic domain that may be modified by phosphorylation [43]or protein interaction. As a result, most CDPKs are mem-brane anchored (Table 1) and the reversibility of the secondsignal may allow CDPKs to shuttle between membranesand the cytosol or nucleus. Diverse cellular localizations of




CDPKs have been observed (Table 1), including the cytosol,nucleus, plasma membrane, endoplasmic reticulum (ER),tonoplast, mitochondria, chloroplasts, oil bodies and per-oxisomes [17,27,34,36,38–40,42,44,46–48], indicating thatCDPKs have access to a plethora of potential substratesthroughout the cell. Moreover, stress-induced nuclear ac-cumulation has been observed for the ice plant (Mesem-bryanthemum crystallinum) McCPK1 [43,49] and thegroundnut (Arachis hypogaea) AhCPK2 [24].

Substrate specificity

Biochemical analyses based on known substrates haveidentified four distinct motifs that define CDPK targetsites [2]. An extensive survey of 534 synthetic peptidestested for in vitro kinase assays with four recombinantArabidopsis CDPKs (AtCPK1, AtCPK10, AtCPK16 andAtCPK34) revealed that CDPK specificity relies eitheron the substrate itself or on kinetic parameters for acommon substrate [25]. This could result from differentCa2+ affinities that can affect substrate accessibility, assuggested by the crystal structure of apicomplexanCDPKs. Indeed, the N-lobe of the CaM-LD is located nearthe N-terminus of the kinase domain in resting conditionswhile the entire CaM-LD rotates around the kinase do-main to release the substrate-binding site in the Ca2+-bound form [19]. Moreover, a domain-swap analysis be-tween NtCDPK1 and AtCPK9 demonstrates the key role ofthe N-terminal variable domain in controlling substratespecificity [50].

Identifying in vivo substrates is an important challengeto unravel CDPK functions. To date, most substrates havebeen described using in vitro assays and are involved indiverse cellular processes, such as primary and secondarymetabolism, stress responses, ion and water transport,transcription and signaling [2,4,10,25]. Interestingly, ananalysis of 274 synthetic peptides harboring in vivo-mapped phosphorylation sites showed a 27% match withAtCPK substrates in an in vitro kinase reaction, suggest-ing potential biological relevance [25]. Yeast-two-hybridscreens with CDPK variants exhibiting altered kinaseactivity have facilitated the identification of putative sub-strates that require in vivo confirmation [49,51,52]. Sever-al transcription factors, including ABF4 (ABA-responsiveelement-binding factor 4), RSG (repression of shootgrowth) and HsfB2a (heat shock factor B2a), have beenfurther characterized as in vivo CDPK substrates involvedin ABA [47], gibberellin [26,50] and herbivore-inducedsignaling [23], respectively. Recently, an in planta randomscreen coupling bimolecular fluorescence complementationand flow cytometry has identified new AtCPK3 interactorsthat could not be revealed by yeast-two-hybrid assays,suggesting the potential to identify plant-specific proteininteractions [53]. Future challenges include correlatingthe mutant phenotypes of particular CDPKs and theircorresponding substrates to establish their biologicalsignificance.

In summary, the multigene family of CDPKs encodes keyCa2+ sensor protein kinases that differ by their expressionpattern, sub-cellular localization, substrate specificities,Ca2+ sensitivities and regulation by lipids, phosphorylationand protein interactions. This huge diversity in molecular

and biochemical properties of CDPKs is likely to providefunctional specificity and redundancy in mediating plantCa2+ signaling in immune and stress responses.

CDPKs in immune signalingPlants sense potential pathogens through the recognitionof microbe-associated molecular patterns (MAMPs) by cell-surface pattern-recognition receptors (PRRs) or effectorproteins via intracellular nucleotide-binding leucine-richrepeat (NB-LRR) immune sensors to initiate overlappingand distinct signaling cascades, including protein kinaseactivation, Ca2+ influx, hormone biosynthesis, oxidativeburst and transcriptional reprogramming. Recent studieshave provided compelling evidence for the involvement ofCDPKs in most of these signaling events (Figure 2).

Hormonal signaling and gene regulation in plant

defense

The tobacco NtCDPK2 from subgroup I is the first CDPKidentified for its role in race-specific plant defense. Theextracellular Avr9 fungal effector strongly activatesNtCDPK2 in tobacco leaves expressing the correspondingtomato (Lycopersicon esculentum) resistance gene Cf9 as aLRR receptor-like protein on the plasma membrane [16].This activation requires both NtCDPK2 autophosphoryla-tion and phosphorylation by an upstream kinase, revealingthe complexity of fine-tuning defense signaling cascades[30]. Transient expression of constitutively activeNtCDPK2 triggers jasmonic acid (JA) and ethylene (ET)accumulation, and subsequent induction of JA- and ET-regulated genes [54]. ET production could occur throughstabilizing the rate-limiting enzyme ACC synthase (ACS)of ET biosynthesis by direct phosphorylation, as observedfor the closest homolog LeCDPK2 in tomato [55] andpurified maize CDPKs [56]. Interestingly, constitutivelyactive NtCDPK2 blocks the Avr9–Cf9 activation of mito-gen-activated protein kinases (MAPKs) in an ET-depen-dent manner, potentially serving as a negative feedback toreset the system after elicitation [54] (Figure 2). In Arabi-dopsis, overexpressing the closest homolog AtCPK1 con-fers broad-spectrum resistance to bacteria and fungi [48].However, unlike NtCDPK2, which reduces salicylic acid(SA) levels and the expression of SA-regulated pathogene-sis-related genes (PR1a and PR2a) [54], long-term AtCPK1overexpression triggers SA accumulation through the in-duction of SA regulatory and biosynthesis genes, PAD4( phytoalexin-deficient 4) and SID2/ICS1 (SA induction-deficient 2/isochorismate synthase 1), and consequentlySA-regulated genes without affecting JA or ET [48]. Inter-estingly, AtCPK1 specifically phosphorylates phenylala-nine ammonia-lyase (PAL) in vitro, which is a keyenzyme in plant defense notably involved in an alternativepathway to produce SA [57]. Although the role of PALphosphorylation in defense is not clear, this finding furthersupports the notion that AtCPK1 plays a crucial role in SAaccumulation (Figure 2). Although we could expect thatprotein kinase orthologs play similar roles in various plantspecies, these results suggest that some closely relatedCDPK homologs might have evolved independently indifferent plant species or in different biological contexts.However, it is also crucial to distinguish transient and

33

Ca2+

channel Ca2+

channel

NB-LRRs

Herbivore a�ack

?

PDF1.2

Wounding HAEs

An�herbivore molecules

RBOH TF

An�microbial chemicals and pep�des,hormones, enzymes and receptors

ROS Camalexinaccumula�on

SA

Ca2+

channel

Transient CDPK ac�va�on

Synergis�ceffectNHL10

MAPKdominantCYP81F2

? Ca2+

MKKKs

MAPKspecific

FRK1

MAPK-CDPK CDPK

specificPHI1

Gene regula�on

Chemicalsand enzymes

Sustained CDPK ac�va�on

Anionchannel

AtCPK4/11CPK5/6

AtCPK1MKK4,5

MPK3,6

MAMPs

PTF TF TF TF

P P P P P P

Pathogen

Effectors Ca2+

MKKKs

MKKs

MPKs

PPAL

Gene regula�on

SID2PAD4

PAD3

RLKs Cf9

Avr9

PRBOH

ROS

NtCDPK2

StCDPK4CDPK5

PACS

P

ETJA

Gene regula�on

Chemicals and enzymes

MKKKs

MKKs

Cell death - HR

PR

Ca2+

AtCPK3

LeCDPK2 Le

CPK1

PACS

P

ET

HsfB2a P

H+

ATPase

P

H+ efflux

Gene regula�on

Early gene regula�on

?

P

K K K

LRR LysM Lec

AtCPK13

SIPK,WIPK NaCDPK4CDPK5

JA

K

RLK

K


Figure 2. CDPK signaling network in immune responses. Microbe-associated molecular pattern (MAMP) perception by different cell-surface receptor kinases (RLKs) with

distinct extracellular domains triggers transient CDPK activation to regulate transcription factors and early gene expression either independently or in coordination with

MAPK cascades. Several CDPKs also activate NADPH oxidases (respiratory burst oxidase homologs, RBOHs) to induce early reactive oxygen species (ROS) production. By

contrast, the sustained CDPK activation by extracellular (Avr9) or intracellular effector proteins leads to biosynthesis of salicylic acid (SA), jasmonic acid (JA) and ethylene

(ET) through regulatory gene induction or enzyme activation such as phenylalanine ammonia-lyase (PAL) and ACC synthase (ACS). CDPKs also trigger a prolonged

oxidative burst involved in cell death and hypersensitive response (HR). Constitutively active NtCDPK2 inhibits MAPK [salicylic acid-induced protein kinase (SIPK) and

wound-induced protein kinase (WIPK)] activation by Avr9–Cf9 in an ET-dependent manner. Herbivores can be sensed through wounding or herbivore-associated elicitors

(HAEs) by unknown receptors to activate MAPKs and Ca2+ influx. The coregulation of ACS by MAPKs and CDPKs leads to ET production, whereas LeCPK1 inhibits the

plasma membrane H+-ATPase to induce extracellular alkalinization. AtCPK3 and AtCPK13 mediate herbivore-induced gene expression by phosphorylating the transcription

factor HsfB2a whereas only AtCPK3 negatively regulates Ca2+ channels. NaCDPK4 and NaCDPK5 negatively regulate defense against herbivores by inhibiting JA

accumulation and subsequent production of defense metabolites. Abbreviations: MKKs, mitogen-activated protein kinase kinases; MKKKs, mitogen-activated protein

kinase kinase kinases; MPKs, mitogen-activated protein kinases; TF, transcription factor.


long-term CDPK overexpression and activation, whichmay lead to distinct direct and indirect physiological con-sequences.

In Arabidopsis leaf cells, the bacterial MAMP flg22 (a22-amino acid peptide of flagellin) transiently activatesmultiple CDPK activities. Interestingly, a cell-based func-tional genomic screen with 25 constitutively active AtCPKsusing a flg22-responsive reporter NHL10-LUC (NDR1/HIN1-like10-luciferase) identified four related CDPKsfrom subgroup I, AtCPK4, AtCPK5, AtCPK6 andAtCPK11, as early transcriptional regulators in MAMPsignaling [17]. Unlike NtCDPK2, these four AtCPKs do notaffect MAPK activation by flg22. Unexpectedly, CDPKsand MAPK cascades differentially regulate flg22-inducedearly genes in at least four regulatory programs, displayingCDPK-specific, MAPK-specific, CDPK/MAPK parallel orCDPK/MAPK synergistic regulation, which imply indepen-dent or co-regulation of common targeted transcriptionfactors, transcription machinery and/or chromatin remo-deling complexes [58,59] (Figure 2). Correlated withgenome-wide analysis of redundant and specific targetgenes modulated by multiple MAMPs and by transientlyexpressed active AtCPK5 and AtCPK11, cpk5 cpk6 double

34

mutant, cpk5 cpk6 cpk11 triple mutant and cpk4 cpk5 cpk6cpk11 quadruple mutant exhibit gradually reduced flg22responsiveness for gene expression and pathogen resis-tance, demonstrating the key positive roles of these CDPKsin convergent MAMP signaling [17].

In summary, various CDPKs from subgroup I mediatetransient and sustained transcriptional reprogramming inplant innate immune responses, and play key roles in theregulation of SA, ET and JA hormonal signaling.

Oxidative burst and cell death

Transient expression of active NtCDPK2 also induces ROS(reactive oxygen species) production and HR (hypersensi-tive response)-like cell death upon exposure to a non-symptom-producing stress [54]. Consistently, silencingits orthologs in Nicotiana benthamiana reduces the HRelicited by the Avr4–Cf4 and Avr9–Cf9 interactions [16].Interestingly, the closest homolog AtCPK1 also triggersROS production by stimulating the NADPH oxidase activ-ity in tomato protoplasts [60]. Active forms of potato(Solanum tuberosum) StCDPK4 and StCDPK5, closehomologs of AtCPK5/AtCPK6, induce ROS production bydirectly phosphorylating the NADPH oxidase RBOHB


(respiratory burst oxidase homolog B) in vivo at the sitetargeted by infection with Phytophthora infestans [61]. Asa consequence, transgenic potato plants overexpressingthe active variant of StCDPK5 display increased ROSproduction, HR-like cell death and resistance to the hemi-biotrophic pathogen P. infestans, but higher susceptibilityto the necrotrophic pathogen Alternaria solani [62]. Coher-ently, the Arabidopsis cpk5 cpk6 double mutant, cpk5 cpk6cpk11 triple mutant and cpk4 cpk5 cpk6 cpk11 quadruplemutant show reduced early ROS production in response toflg22 [17]. These results suggest that multiple CDPKs fromsubgroup I play a key role in the defense-induced oxidativeburst by activating NADPH oxidases through direct phos-phorylation (Figure 2). Homologs of AtCPK5/AtCPK6 alsomediate plant defense in monocots. The rice OsCPK13induces cell death, accumulation of PR proteins and up-regulation of some defense genes when ectopicallyexpressed in sorghum (Sorghum bicolor) [63], whereasthe active form of barley (Hordeum vulgare) HvCDPK4triggers cell death [64]. Thus, CDPKs display conserveddefense functions among plant species in promoting celldeath. However, genetic manipulation of CDPKs in cropprotection may need specifically tailored strategies consid-ering the site and duration of CDPK activation and thetargeted pathogens.

By contrast, several CDPKs have been shown to playnegative roles in plant defense. In barley, HvCDPK3 fromsubgroup II promotes host cell entry of the powdery mildewfungus during both compatible and incompatible interac-tions [64]. In rice, overexpressing OsCPK12 from subgroupII confers susceptibility to both virulent and avirulent blastfungus, potentially through ABA hypersensitivity and re-duction in ROS production [65]. Interestingly, Ca2+- andCaM-regulated protein kinase (CCaMK) is a central sig-naling hub in root nodule and arbuscular mycorrhizasymbioses in plants [66]. However, plant defense mustbe shut down in the initial phase of the plant–microbeinteraction, so that the symbiont is not recognized as apathogen. In Medicago truncatula, MtCDPK1 from sub-group IV is likely to be involved in this process because theMtCDPK1-silenced plants are compromised in establish-ing symbiotic interactions and display enhanced ROS pro-duction induced by Nod factors and increased expression ofcell wall biosynthesis and defense genes [67].

Responses to wounding and herbivore attacks

During insect attack, wounded tissues constitute entrysites for herbivory elicitors, making wounding perceptiona part of herbivore recognition. A recent screen of 19 cpkT-DNA insertion mutant lines identified two CDPKs,AtCPK3 and AtCPK13, as positive regulators of PDF1.2induction by Spodoptera littoralis caterpillars [23]. Thisregulation probably occurs through phosphorylation-de-pendent activation of the transcription factor HsfB2a,without affecting the level of ET, JA or ABA. Moreover,AtCPK3, but not AtCPK13, also triggers a negative feed-back on herbivore-induced Ca2+ signals, indicating thatCDPKs can play redundant as well as specific functions inplant defense. Given that AtCPK3 can be activated by flg22in protoplasts [44] and induce the flg22-responsive geneNHL10 [17], it may also be involved in MAMP signaling.

By contrast, the tomato LeCDPK2 phosphorylates theethylene biosynthesis enzyme LeACS2 at the site phos-phorylated in vivo after wounding, suggesting thatLeCDPK2 contributes to ethylene production in responseto wounding [55]. Interestingly, MAPKs also phosphory-late LeACS2 at a different site, and both CDPK and MAPKphosphorylations are required simultaneously to stabilizeLeACS2 in vivo [55]. Wounding also induces extracellularalkalinization through the inhibition of the plasma mem-brane H+-ATPase, which may be mediated in tomato by themembrane-anchored LeCPK1 [12] (Figure 2). In maize,ZmCPK11, which is closely related to AtCPK4/AtCPK11,is activated by wounding in a JA-dependent pathway;however, its precise biological function remains to be de-termined [68]. Recently, two redundant CDPKs from sub-group IV in coyote tobacco (Nicotiana attenuata),NaCDPK4 and NaCDPK5, were proposed to negativelyregulate herbivore resistance by blocking JA and defensemetabolite accumulation, without affecting the expressionof JA biosynthesis enzymes [69].

Thus, various CDPKs from different subgroups mediateplant responses to wounding and herbivores through theregulation of hormone biosynthesis, such as ET and JA,and gene expression, either with a positive or a negativeeffect.

CDPKs in hormonal and abiotic stress signalingGene regulation in ABA, drought and salt stress

signaling

ABA is a key stress hormone that mediates plant responsesto drought and salinity. AtCPK10 and AtCPK30 from sub-group III are the first CDPKs identified as positive regula-tors of the barley stress- and ABA-inducible HVA1 promoterin maize protoplasts [15]. Constitutively active AtCPK10also induces endogenous stress- and ABA-inducible genes inArabidopsis leaf cells (Y. Niu and J. Sheen, unpublished),suggesting a conservation of specific CDPK functions indicots and monocots. This transcriptional induction islikely to occur through the ABA-responsive transcriptionfactors, ABFs, which were identified as in vitro substratesfor several CDPKs from subgroups I and III [39,47](Figure 3). In particular, AtCPK32 activates ABF4 in vivo,resulting in the induction of ABF4 target genes [47].AtCPK4 and AtCPK11 regulate both ABF1 and ABF4and induce gene expression several hours after ABA elici-tation, suggesting a role in long-term adaptation [39]. Thismodified gene expression correlates with ABA hypersensi-tivity and increased tolerance to drought and salt in plantsoverexpressing AtCPK4 or AtCPK11, whereas the cpk4cpk11 double mutant exhibits the opposite phenotypes.Given that only selected ABA-responsive genes are partiallyaffected in the cpk4 cpk11 double mutant, other regulatorsalso contribute to ABA signaling [39]. Similar results wereobserved in rice transgenic plants overexpressing OsCPK21[70] and OsCPK13 (OsCDPK7) [71], suggesting that ABA-induced transcriptional reprogramming via ABFs is likelyto be a key feature of CDPK signaling in both monocotsand dicots.

Although the role of CDPK phosphorylation has notbeen thoroughly investigated, other transcription factorsinvolved in stress-induced gene regulation have also been

35

Salinity Drought Cold

Na+ toxicity Osmo�c stress

AtCPK1/2?

AtCPK3

AtCPK23

OsCPK12

ACA2

Ca2+

signalK+

uptakeROS

APX8 RBOHI

Gene regula�on

AtCPK6

AtCPK21

Prolineaccumula�on

AtCPK12

McCPK1

SoCDPK

Watertransport

AQP

Gene regula�on Stomatal closure

CSP1

OsCPK13CPK21

ABA

AtCPK4

CPK11

AtCPK30CPK32

AtCPK10

AtDi19s

ABIs

SnRK2sPABFs

PPPPP

ACPK1Gm

CDPK

AtCPK3CPK6

AtCPK21CPK23

K+inwardchannel

KAT1

SnRK2s

Anionchannel

Ca2+

channelSLAC1SLAH3

ABIs

P P P

AtCPK1

OsCPK13

OsCPK7

Calre�culinaccumula�on

Cold tolerance

? ?

AtCPK16


Figure 3. CDPK signaling network in abiotic stress responses. Plants sense drought and salinity through Na+ toxicity, osmotic stress and ABA synthesis to activate CDPKs, which

regulate K+ uptake, ROS production, accumulation of compatible osmolytes (proline), water transport (aquaporin, AQP) and gene expression. Redundant CDPKs modulate gene

expression by activating the transcription factors ABFs and AtDi19s. AtCPK12 is a negative regulator that stimulates the protein phosphatase ABIs, inhibiting SnRK2- and CDPK-

dependent transcriptional regulation. CDPKs also promote stomatal closure by inhibiting the K+ inward channel (KAT1), and activating slow-type anionic channels (SLAC1 and

SLAH3). CDPKs and SnRK2s share common substrates (ABFs and channels) and common downregulators (ABIs). Some CDPKs also inhibit permeable Ca2+ channels or Ca2+-

pumps (ACA2) in a negative feedback loop. Several CDPKs have been shown to trigger cold tolerance; however, the molecular mechanism is not understood.


identified as CDPK substrates in vitro. They include theArabidopsis dehydration-inducible gene family AtDi19,which encodes nuclear zinc-finger proteins [25,51], andthe ice plant pseudo-response regulator transcription fac-tor CSP1 [49] (Figure 3). By contrast, AtCPK12, the closesthomolog of AtCPK4/AtCPK11, inhibits ABA responses bystimulating the negative regulator ABI2 (ABA insensitive2), although the role of ABI2 phosphorylation by AtCPK12requires further studies [72].

Metabolic and transport regulation in drought and salt

stress signaling

Drought and salinity trigger the production of ROS, whichmust be detoxified. Soybean GmCDPKa and GmCDPKg

have been shown to phosphorylate in vitro the serineacetyltranferase 2;1 (GmSerat2;1) involved in cysteinebiosynthesis, at the site phosphorylated in vivo after oxi-dative stress [73]. Because the phosphorylation releasesthe feedback inhibition by cysteine, CDPKs may partici-pate in anti-oxidant responses by providing cysteine forglutathione production. In rice, OsCPK12 regulates ROShomeostasis under salt stress conditions by inducing theexpression of ROS scavenger genes OsAPX2/OsAPX8 andrepressing the NADPH oxidase gene OsRBOHI, leading toincreased salt tolerance [65]. Overexpressing AtCPK6 con-fers drought tolerance, correlated with enhanced geneexpression and accumulation of the compatible osmolyteproline, but reduces lipid peroxidation, probably throughdecreased ROS production [74]. However, the cpk6 singlemutant does not exhibit any stress phenotype, as observedin pathogen responses because of functional redundancyamong CDPKs [17]. By contrast, AtCPK21 is a negativeregulator of osmotic responses and inhibits proline accu-mulation [18]. The subtle enhanced drought tolerance inthe cpk21 mutant is likely to result from the compensatedoverexpression of the closest homolog AtCPK23 [18], whichalso negatively regulates drought and salt resistance by

36

inhibiting K+ uptake [75] (Figure 3). Interestingly, unlikein defense responses [17,23], AtCPK3 does not regulategene expression in salt stress signaling but modulates thephosphoproteome independently of MAPK cascades [44].In particular, two putative substrates, a glutathione-S-transferase and a subunit of a potassium channel, suggesta role of AtCPK3 in anti-oxidant responses and K+ uptake,respectively.

Limiting water loss is crucial during dehydration con-ditions and may occur through the downregulation ofaquaporin water channels, as observed in spinach (Spina-cea oleracea) with the CDPK-stimulated aquaporin PM28A[76,77]. Regulating Ca2+ signals is also a key component ofstress signaling to ensure that specific responses are in-duced by each stress. The Arabidopsis auto-inhibited cal-cium ATPase ACA2 has been shown to play a crucial role ingenerating the appropriate Ca2+ signal in yeast exposed tohigh salinity [78]. Interestingly, the active form of AtCPK1inhibits the basal activity of ACA2 and blocks stimulationby CaM in yeast [79]. However, AtCPK1 is not localized inthe ER, unlike its closest homolog AtCPK2 and ACA2,suggesting that AtCPK2 might regulate ACA2 in planta[40,48]. Thus, multiple CDPKs differentially modulatetranscription factors, metabolic enzymes, ion and watertransport to positively or negatively regulate drought andsalt responses.

Regulation of stomatal movements

Ca2+ is an essential component of guard cell signaling, andregulates ion fluxes involved in stomatal movements, no-tably through CDPK-dependent phosphorylation of chan-nels. The cpk3 cpk6 double mutant is impaired in ABA-activation of slow-type anionic channels and Ca2+ perme-able channels, resulting in decreased ABA-induced stoma-tal closure [80]. Despite a weak in vivo interaction with themajor guard cell Cl– channel SLAC1 [22], AtCPK6 but notAtCPK3 was recently shown to phosphorylate SLAC1 at


S59 and to activate the channel in Xenopus (Xenopuslaevis) oocytes [81]. Similarly, two close homologs fromsubgroup II, AtCPK21 and AtCPK23, also phosphorylateand activate SLAC1 and its related NO3

– channel SLAH3in response to ABA [22,82]. AtCPK23 preferentially reg-ulates SLAC1 independently of Ca2+ whereas AtCPK21mainly regulates SLAH3 in a Ca2+-dependent manner(Figure 3). Whether the three CDPKs target the samephosphosite remains to be determined to understand thebiological relevance of such a redundancy. Surprisingly,cpk21 and cpk23 single mutants do not exhibit any stomataphenotype, unlike cpk3 and cpk6 [22,80,82]. It is thuspossible that the stronger phenotypes observed in cpk3and cpk6 result from the inhibition of Ca2+ currents thatwould impact global Ca2+-regulated responses rather thanonly inhibiting anionic channels. Interestingly, unlike oth-er cpk mutants, cpk6 is also impaired in methyl jasmonate(MeJA)-induced S-type anionic current and stomatal clo-sure [83]. Because MeJA and ABA differentially mediatepathogen and abiotic stress responses, distinct hormonalpathways may regulate stomatal closure upon biotic andabiotic stresses.

The cpk4 cpk11 double mutant is also partially compro-mised in ABA-induced stomatal closure with enhanced leafwater loss [39], whereas its closest homolog in grapevine(Vitis vinifera) ACPK1 confers the opposite phenotypewhen overexpressed in Arabidopsis [84]. The inhibitionof K+ inward channels, such as KAT1, also contributes tostomatal closure. In the broad bean (Vicia faba), a CDPKphosphorylates KAT1 in vitro [85], which inhibits thechannel activity [86]. Moreover, the ABA inhibition ofK+ inward channels is abolished in the Arabidopsiscpk10 mutant, leading to reduced stomatal closure anddrought hypersensitivity, suggesting that AtCPK10 maydownregulate KAT1 in vivo [27]. AtCPK1 stimulates avacuolar Cl– channel, resulting in Cl– uptake into thevacuole and stomatal opening [87]. Despite some discre-pancies in ABA-induced stomatal closure in some cpkmutants, the impaired Ca2+-induced stomatal closure inmultiple cpk mutants from subgroups I, II and III, cpk4cpk11 and cpk3 cpk6 double mutants, cpk10 single mutantand cpk7 cpk8 cpk32 triple mutant, demonstrates thecrucial role of CDPKs in regulating stomatal movements,even though the molecular mechanism remains to beelucidated [88].

Cold tolerance

Ca2+-mediated early cold induction of the key transcription-al regulators, the CBFs (cold-responsive element-bindingfactors), is crucial for cold tolerance. Several CaM-interact-ing transcription activators (CAMTAs) have been shown tobind to the CBF2 promoter and the camta3 mutant isimpaired in cold-induction of CBF1 and CBF2. The reducedfreezing tolerance of the camta1 camta3 double mutantfurther supports the notion that CAMTAs play a role incold-stimulated Ca2+ signaling [89]. However, the functionsof CDPKs in cold stress signaling remain mostly elusive.Unlike in salt or drought signaling, OsCPK13 (OsCDPK7)confers cold resistance without affecting transcriptionalregulation [71]. Similarly, AtCPK1 does not affect CBFinduction but regulates the phosphoproteome after cold

treatment [90]. A membrane-bound rice CDPK is activatedby cold after 18–24 h, suggesting a role in the adaptiveprocess rather than early responses [91]. Likewise, over-expressing OsCPK7 (OsCDPK13), which is preferentiallyexpressed in cold-tolerant rice varieties, triggers cold resis-tance and the accumulation of the cold-responsive chaper-one calreticulin [37,92]. Thus, CDPKs are clearly involved incold tolerance, but their molecular functions remain to beexplored (Figure 3).

CDPKs at the crossroad of stress signaling networksRecent advances have revealed CDPKs as central regula-tors of Ca2+-mediated immune and stress responses thatare crucial for plant survival. Some key players, includingAtCPK1, AtCPK3, AtCPK4, AtCPK6, AtCPK11, OsCPK12and OsCPK13, represent crucial signaling nodes that me-diate plant responses to both abiotic stress and pathogens(Table 1). Beside some specificity, several CDPKs alsoshow functional redundancy that may provide robust plantresponsiveness and adaptability to adverse environmentalconditions. For instance, AtCPK4, AtCPK5, AtCPK6 andAtCPK11 co-regulate gene expression in early MAMPsignaling [17], whereas AtCPK4, AtCPK11, AtCPK10,AtCPK30 and AtCPK32 all mediate ABA responsesthrough ABFs [15,39,47]. There is also functional redun-dancy with other protein kinase families such as the SNF1-related kinases 2 (SnRK2s) [93,94] and MAPKs [17] (Fig-ures 2 and 3). In particular, several effector proteins ofguard cell signaling such as KAT1, SLAC1, RBOHs andABFs are targeted by both CDPKs and SnRK2s, potential-ly at different sites, although the coregulation has not beeninvestigated [95]. Furthermore, analogous to the roles inantagonizing SnRK2s [95], the protein phosphatase 2C(PP2C) ABI1, ABI2 and/or related PP2Cs inhibit CDPKsin stress and ABA signaling, such as AtCPK10/AtCPK30-mediated gene regulation [15,96] or AtCPK21/AtCPK23activation of anionic channels [22,82].

More complex interactions have been observed betweenCDPKs and MAPK cascades, from synergism [17,55] toindependence [44] and antagonism [54]. The multiplecrosstalks between CDPKs and SnRK2s or MAPKs provideadditional layers of regulation to fine-tune plant immuneand stress responses. Strikingly, some CDPKs may playopposite roles in different cell types. For instance,AtCPK21/AtCPK23 activate SLAC1/SLAH3 in guard cellsto promote stomatal closure [22,82], whereas cpk21 andcpk23 mutants are drought tolerant at the whole plantlevel [18,75]. Distinct CDPK substrates are likely to beresponsible for different physiological functions in diversecellular contexts.

Concluding remarks and perspectivesExtensive research efforts with integrative approaches haveprovided conclusive evidence that CDPKs are versatile andevolutionarily conserved Ca2+-sensors/transducers thatfunction in a diverse array of plant processes in responseto environmental challenges. Combined with expressionpatterns, intracellular localizations/translocations andmodulation by lipids, phosphorylation and interacting pro-teins, the broad ranges of Ca2+ sensitivity and substratespecificity of CDPKs dictate complex and sophisticated Ca2+

37


signaling networks via protein phosphorylation to coordi-nate the dynamic plant cellular processes. Future molecu-lar, cellular, genetic, genomic and phosphoproteomicstudies should lead to more precise understanding of specificand redundant roles of CDPKs in the immune and stresssignaling networks in cooperation with other Ca2+ sensorsand protein kinases, such as RLKs (receptor-like kinases),MAPKs, SnRK1s, SnRK2s and SnRK3s/CIPKs (CBL-inter-acting protein kinases) [5–7,9,17,25,88,93,94,97,98]. Con-sidering that Ca2+ signals are restricted and localized insidethe cells, establishing molecular, cellular and genetic linkswith specific Ca2+ channels, pumps and transporters [8] in aspatio-temporal analysis of CDPK activation and transloca-tion is crucial to decipher Ca2+-mediated signaling networksin plants. Unlike the Ca2+-independent SnRK2s andMAPKs, analyzing the in vivo activation of CDPKs has beenlimited because of the difficulty in maintaining precise Ca2+

levels reflecting the physiological states in cell extracts.Developing specific anti-phosphopeptide antibodies raisedagainst active CDPKs or their immediate substrates shouldfacilitate the monitoring of CDPK activation in planta[17,25,30,99,100]. A complementary genetic approach bymutating the EF-hands should establish a functional linkbetween CDPKs and Ca2+ signaling [18,36]. Finally, identi-fying in vivo substrates and the unique regulatory featuresof each isoform should provide new insights into CDPKsignaling to understand their integrated roles in diversebiological responses, a prerequisite for genetic manipulationof agronomically valuable traits.

Disclosure statementThe authors declare no conflict of interest.

AcknowledgmentsThe projects on CDPK signaling have been supported by the NationalScience Foundation and the National Institutes of Health (J.S.). M.B. wassupported by a Marie Curie International fellowship within the 6thEuropean Community Framework Program.

Appendix A. Supplementary dataSupplementary data associated with this article can befound, in the online version, at doi:10.1016/j.tplants.2012.08.008.

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CDPKs in immune and stress signaling - Molbio